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1.  Co-grafting of neural stem cells with olfactory en sheathing cells promotes neuronal restoration in traumatic brain injury with an anti-inflammatory mechanism 
Background
We sought to investigate the effects of co-grafting neural stem cells (NSCs) with olfactory ensheathing cells (OECs) on neurological behavior in rats subjected to traumatic brain injury (TBI) and explore underlying molecular mechanisms.
Methods
TBI was established by percussion device made through a weight drop (50 g) from a 30 cm height. Cultured NSCs and OECs isolated from rats were labeled by Hoechst 33342 (blue) and chloromethyl-benzamidodialkyl carbocyanine (CM-Dil) (red), respectively. Then, NSCs and/or OECs, separately or combined, were transplanted into the area surrounding the injury site. Fourteen days after transplantation, neurological severity score (NSS) were recorded. The brain tissue was harvested and processed for immunocytochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reverse transcription-polymerase chain reaction (RT-PCR).
Results
Significant neurological function improvement was observed in the three transplant groups, compared to the TBI group, and co-transplantation gave rise to the best improvement. Morphological evaluation showed that the number of neurons in cortex from combination implantation was more than for other groups (P <0.05); conversely, the number of apoptotic cells showed a significant decrease by TUNEL staining. Transplanted NSCs and OECs could survive and migrate in the brain, and the number of neurons differentiating from NSCs in the co-transplantation group was significantly greater than in the NSCs group. At the molecular level, the expressions of IL-6 and BAD in the co-graft group were found to be down regulated significantly, when compared to either the NSC or OEC alone groups.
Conclusion
The present study demonstrates for the first time the optimal effects of co-grafting NSCs and OECs as a new strategy for the treatment of TBI via an anti-inflammation mechanism.
doi:10.1186/1742-2094-11-66
PMCID: PMC3977666  PMID: 24690089
Neural stem cells (NSCs); Olfactory en sheathing cells (OECs); Traumatic brain injury (TBI); Anti-inflammation
2.  OECs transplantation results in neuropathic pain associated with BDNF regulating ERK activity in rats following cord hemisection 
BMC Neuroscience  2013;14:80.
Background
The olfactory ensheathing cells (OECs) derived from olfactory bulb (OB) may improve motor function after transplantation in injured spinal cord. However, the effects of OEC transplantation on sensory function have not been reported yet. The purpose of this study is to investigate whether OEC transplantation could affect the sensory function and to analyze the underlying mechanism.
Results
OEC transplantation into the hemisected spinal cords can result in hyperalgesia, indicated by radiant and mechanical stimuli towards the plantar surface in rats. This could be associated with upregulation of Brain Derived Neurotrophic Factor (BDNF), indicated by RT-PCR. Immunofluorecent staining showed that BDNF was mainly located in the neurons of the laminas I and II of the dorsal horn. Moreover, a notable upregulation on the level of p-ERK (phosphorylation of extracellular signal-regulated kinase), the downstream molecule of BDNF, was detected by using Western Blot. These findings indicate that the increased BDNF level associated with the p-ERK was possibly involved in neuropathic pain in hemisected spinal cord subjected to OEC transplantation.
Conclusions
The transplantation of OECs may induce the noticeable pain hypersensitivity in rats after hemisected spinal cord injury, and the possible mechanism may be associated with the phosphorylation of ERK and the activated BDNF overexpression.
doi:10.1186/1471-2202-14-80
PMCID: PMC3734147  PMID: 23914898
Olfactory ensheathing cells; Spinal cord injury; Hemisection; Cell transplantation; Rat; p-ERK; BDNF; Hyperalgesia
3.  Genes Influenced by the Non-Muscle Isoform of Myosin Light Chain Kinase Impact Human Cancer Prognosis 
PLoS ONE  2014;9(4):e94325.
The multifunctional non-muscle isoform of myosin light chain kinase (nmMLCK) is critical to the rapid dynamic coordination of the cytoskeleton involved in cancer cell proliferation and migration. We identified 45 nmMLCK-influenced genes by bioinformatic filtering of genome–wide expression in wild type and nmMLCK knockout (KO) mice exposed to preclinical models of murine acute inflammatory lung injury, pathologies that are well established to include nmMLCK as an essential participant. To determine whether these nmMLCK-influenced genes were relevant to human cancers, the 45 mouse genes were matched to 38 distinct human orthologs (M38 signature) (GeneCards definition) and underwent Kaplan-Meier survival analysis in training and validation cohorts. These studies revealed that in training cohorts, the M38 signature successfully identified cancer patients with poor overall survival in breast cancer (P<0.001), colon cancer (P<0.001), glioma (P<0.001), and lung cancer (P<0.001). In validation cohorts, the M38 signature demonstrated significantly reduced overall survival for high-score patients of breast cancer (P = 0.002), colon cancer (P = 0.035), glioma (P = 0.023), and lung cancer (P = 0.023). The association between M38 risk score and overall survival was confirmed by univariate Cox proportional hazard analysis of overall survival in the both training and validation cohorts. This study, providing a novel prognostic cancer gene signature derived from a murine model of nmMLCK-associated lung inflammation, strongly supports nmMLCK-involved pathways in tumor growth and progression in human cancers and nmMLCK as an attractive candidate molecular target in both inflammatory and neoplastic processes.
doi:10.1371/journal.pone.0094325
PMCID: PMC3979809  PMID: 24714365
4.  Heterozygous alleles restore male fertility to cytoplasmic male-sterile radish (Raphanus sativus L.): a case of overdominance 
Journal of Experimental Botany  2013;64(7):2041-2048.
The practice of hybridization has greatly contributed to the increase in crop productivity. A major component that exploits heterosis in crops is the cytoplasmic male sterility (CMS)/nucleus-controlled fertility restoration (Rf) system. Through positional cloning, it is shown that heterozygous alleles (RsRf3-1/RsRf3-2) encoding pentatricopeptide repeat (PPR) proteins are responsible for restoring fertility to cytoplasmic male-sterile radish (Raphanus sativus L.). Furthermore, it was found that heterozygous alleles (RsRf3-1/RsRf3-2) show higher expression and RNA polymerase II occupancy in the CMS cytoplasmic background compared with their homozygous alleles (RsRf3-1/RsRf3-1 or RsRf3-2/RsRf3-2). These data provide new insights into the molecular mechanism of fertility restoration to cytoplasmic male-sterile plants and illustrate a case of overdominance.
doi:10.1093/jxb/ert065
PMCID: PMC3638831  PMID: 23630327
Cytoplasmic male sterility; fertility restoration; heterozygous alleles; overdominance; radish.
5.  Two cases of multiple ossifying fibromas in the jaws 
Diagnostic Pathology  2014;9:75.
Background
The clinicopathologic characteristics of multiple ossifying fibroma (OF) are unclear due to the condition’s rarity, making diagnosis challenging. Sporadic multiple OFs must be distinguished from hyperparathyroidism-jaw tumour syndrome (HPT-JT) related OF and other fibro-osseous lesions.
Methods
Multiple OF cases were identified from ossifying fibroma cases. Clinical data including age, sex, anatomic site, radiographic features, clinical impression, treatment and available follow-up data as well as serum calcium, phosphorus, and parathyroid hormone (PTH) were recorded. GNAS and HRPT2 genetic mutations were examined in the two present cases. Case reports of sporadic multiple ossifying fibroma and HPT-JT-related OF were also reviewed.
Results
The two present cases were confirmed as sporadic multiple OF, with no genetic GNAS and HRPT2 mutations found. The incidence of sporadic multiple ossifying fibroma was 2.0% (2/102). The total 18 sporadic multiform OF cases were characterized as followed: 13 (72.2%) female; 5 (27.8%) male; mean age 28.6 years; 2/16 (11.1%) cases only in the mandible; 4/18 (22.2%) cases only in the maxilla; and 12/18 (66.7%) cases in both the maxilla and mandible. Radiographically, the lesions were radiolucent in 5/18 (27.8%) cases and mixed density in 13/18 (72.2%) cases. Along with 24 cases of HPT-JT related OF were reviewed, sixteen (66.7%) patients were diagnosed with a single lesion, and 8 patients (33.3%) were diagnosed with multiple jaw lesions.
Conclusions
Sporadic multiple OFs are very rare, but must be distinguished from HPT-JT related OF. We strongly recommend that patients diagnosed with multiple ossifying fibromas receive serum PTH testing and mutation screening of HRPT2.
Virtual slides
http://www.diagnosticpathology.diagnomx.eu/vs/1194507146115753
doi:10.1186/1746-1596-9-75
PMCID: PMC3974450  PMID: 24678936
Multiple ossifying fibroma; HPT-JT; Fibrous dysplasia; GNAS gene; HRPT2 gene; Osseous dysplasia
6.  Elevated Interleukin-32 Expression Is Associated with Helicobacter pylori-Related Gastritis 
PLoS ONE  2014;9(3):e88270.
Background
Interleukin-32 (IL-32) is a recently discovered proinflammatory cytokine involved in inflammatory diseases. We investigated the expression of IL-32 and its regulation mechanism in the inflammatory response of patients with Helicobacter pylori (H. pylori) infection.
Design and Methods
IL-32 mRNA and protein expression in gastric tissues was detected by quantitative real-time PCR and immunohistochemistry. The regulation of IL-32 in human gastric epithelia cell line AGS was investigated by different cytokine stimulation and different H. pylori strain infection.
Results
Gastric IL-32 mRNA and protein expression were elevated in patients with H. pylori infection and positively correlated with gastritis. In H. pylori-infected patients, the mRNA level of IL-32 was also correlated with that of proinflammatory cytokines IL-1β and TNF-α. In vitro IL-1β and TNF-α could upregulate IL-32 mRNA and protein level in AGS cells, which was dependent on NF-κB signal pathway. The regulation of IL-32 expression in response to H. pylori-infection could be weakened by using neutralizing antibodies to block IL-1β and TNF-α. Moreover, H. pylori-infected AGS cells also induced IL-32 mRNA and protein expression, which was dependent on CagA.
Conclusions
IL-32 level is elevated in patients with H. pylori infection and its expression is regulated by proinflammatory stimuli, suggesting that IL-32 may play a role in the pathogenesis of H. pylori-related gastritis.
doi:10.1371/journal.pone.0088270
PMCID: PMC3954549  PMID: 24633341
7.  Detection and Analysis of Human Papillomavirus (HPV) DNA in Breast Cancer Patients by an Effective Method of HPV Capture 
PLoS ONE  2014;9(3):e90343.
Despite an increase in the number of molecular epidemiological studies conducted in recent years to evaluate the association between human papillomavirus (HPV) and the risk of breast carcinoma, these studies remain inconclusive. Here we aim to detect HPV DNA in various tissues from patients with breast carcinoma using the method of HPV capture combined with massive paralleled sequencing (MPS). To validate the confidence of our methods, 15 cervical cancer samples were tested by PCR and the new method. Results showed that there was 100% consistence between the two methods.DNA from peripheral blood, tumor tissue, adjacent lymph nodes and adjacent normal tissue were collected from seven malignant breast cancer patients, and HPV type 16(HPV16) was detected in 1/7, 1/7, 1/7and 1/7 of patients respectively. Peripheral blood, tumor tissue and adjacent normal tissue were also collected from two patients with benign breast tumor, and 1/2, 2/2 and 2/2 was detected to have HPV16 DNA respectively. MPS metrics including mapping ratio, coverage, depth and SNVs were provided to characterize HPV in samples. The average coverage was 69% and 61.2% for malignant and benign samples respectively. 126 SNVs were identified in all 9 samples. The maximum number of SNVs was located in the gene of E2 and E4 among all samples. Our study not only provided an efficient method to capture HPV DNA, but detected the SNVS, coverage, SNV type and depth. The finding has provided further clue of association between HPV16 and breast cancer.
doi:10.1371/journal.pone.0090343
PMCID: PMC3948675  PMID: 24614680
8.  Transfer of Intracellular HIV Nef to Endothelium Causes Endothelial Dysfunction 
PLoS ONE  2014;9(3):e91063.
With effective antiretroviral therapy (ART), cardiovascular diseases (CVD) are emerging as a major cause of morbidity and death in the aging HIV-infected population. To address whether HIV-Nef, a viral protein produced in infected cells even when virus production is halted by ART, can lead to endothelial activation and dysfunction, we tested Nef protein transfer to and activity in endothelial cells. We demonstrated that Nef is essential for major endothelial cell activating effects of HIV-infected Jurkat cells when in direct contact with the endothelium. In addition, we found that Nef protein in endothelial cells is sufficient to cause apoptosis, ROS generation and release of monocyte attractant protein-1 (MCP-1). The Nef protein-dependent endothelial activating effects can be best explained by our observation that Nef protein rapidly transfers from either HIV-infected or Nef-transfected Jurkat cells to endothelial cells between these two cell types. These results are of in vivo relevance as we demonstrated that Nef protein induces GFP transfer from T cells to endothelium in CD4.Nef.GFP transgenic mice and Nef is present in chimeric SIV-infected macaques. Analyzing the signal transduction effects of Nef in endothelial cells, we found that Nef-induced apoptosis is mediated through ROS-dependent mechanisms, while MCP-1 production is NF-kB dependent. Together, these data indicate that inhibition of Nef-associated pathways may be promising new therapeutic targets for reducing the risk for cardiovascular disease in the HIV-infected population.
doi:10.1371/journal.pone.0091063
PMCID: PMC3946685  PMID: 24608713
9.  Embryonic oxygen enhances learning ability in hatchling lizards 
Frontiers in Zoology  2014;11:21.
Introduction
Producing smart offspring is an important fitness trait; individuals with enhanced cognitive ability should be more adept at responding to complex environmental demands. Cognitive ability can be influenced by conditions experienced during embryonic development. Although oxygen is necessary for embryonic development, availability can be limited within the nest environment because of substrate type, hydric conditions, and temperature. We do not yet understand, however, whether oxygen availability during embryonic development influences offspring fitness, especially cognitive ability. To address this question we incubated Mongolian Racerunner lizard (Eremias argus) eggs under hypoxic (12% O2), normoxic (21% O2), and hyperoxic conditions (30% O2).
Results
Hypoxia not only slowed hatching time, but also resulted in constrained cognitive ability relative to hatchlings experiencing normoxic or hyperoxic incubation conditions. Oxygen did not influence hatching success, body size or sprint speed of hatchlings.
Conclusions
Oxygen availability during embryonic development has important influences on incubation duration and cognitive ability of hatchling lizards. This study provides the first evidence that oxygen availability during embryonic development can modify cognitive ability of oviparous reptiles.
doi:10.1186/1742-9994-11-21
PMCID: PMC3973888  PMID: 24589451
Embryonic development; Oxygen concentration; Cognitive ability; Mongolian Racerunner lizard; Eremias argus
10.  Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R 
BMC Cancer  2014;14:134.
Background
Resistance to humanized monoclonal erbB2/HER2 antibody, trastuzumab (Herceptin), has become a pivotal obstacle for targeted therapy of HER2-positive breast cancers. The activation of alternative growth factor receptors, in particular, the insulin-like growth factor 1 receptor (IGF1R), represents a common feature of trastuzumab-refractory cells; however, the underlying mechanism remains elusive.
Methods
Trastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3 cells in the presence of trastuzumab. Among the differentially expressed microRNAs (miRNAs) screened by microarray analysis, candidate miRNA(s) predicted to target IGF1R was studied for its role in conferring trastuzumab resistance. The mechanism underlying decreased expression of IGF1R-targeted miRNA in refractory cells was also addressed.
Results
miR-375, which was downregulated and predicted to target IGF1R in trastuzumab-resistant HER2-positive breast cancer cells, could indeed inhibit the cellular luciferase activity in a reporter construct containing the 3′-UTR of IGF1R. Overexpression of miR-375 restored the sensitivity of cells to trastuzumab, while inhibition of miR-375 conferred trastuzumab resistance on HER2-positive breast cancer cells. Blockade of DNA methylation and histone deacetylation restored the expression of miR-375 in trastuzumab-resistant cells. A reverse correlation between the levels of miR-375 and IGF1R was validated in clinical breast cancers.
Conclusions
Epigenetic silencing of miR-375 causes the upregulation of IGF1R, which at least partially underlies trastuzumab resistance of breast cancer cells. Our study has implications for miR-375 as a potential target in combination with trastuzumab for treating HER2-positive breast cancers.
doi:10.1186/1471-2407-14-134
PMCID: PMC3974046  PMID: 24571711
miR-375; Insulin-like growth factor 1 receptor; Trastuzumab resistance; erbB2/HER2; Breast cancer
11.  A clinicopathologic study on calcifying epithelial odontogenic tumor: with special reference to Langerhans cell variant 
Diagnostic Pathology  2014;9:37.
Background
Calcifying epithelial odontogenic tumour (CEOT) is a rare benign odontogenic tumour, and its Langerhans cell variant is even rarer. Due to the limited number of recorded cases, the biological behaviour and histogenesis of the Langerhans cell variant of CEOT are not yet fully understood. Thus, the correlation between conventional CEOT and the Langerhans cell variant remains to be clarified.
Material (cases)
Eight cases of CEOT including 2 cases of Langerhans cell variant were clinicopathologically studied and the English language literature was reviewed. Langerhans cells were detected in 2 cases of conventional CEOT and in 2 cases of Langerhans cell variant by immunohistochemistry.
Results and findings
In the 6 cases of conventional CEOT, 5 tumours involved the premolar and molar region and the anterior portion of the mandible was affected in 1 case. Four patients were followed for 2–7 years and did not show any sign of recurrence. A review of the English language literature revealed 5 cases; combined with the present 2 new cases, a total of 7 cases of Langerhans cell variant of CEOT were collected. The patients were all Asian. Six tumours occurred in the maxilla and 1 in mandible; all mainly involved the anterior region of the jaws. Five patients were followed for 2-10 years and did not show any evidence of recurrence. Langerhans cells can be seen in both the conventional and the Langerhans cell variant of CEOT; however, increased numbers of Langerhans cells are seen in the latter.
Conclusions
Although the Langerhans cell variant of CEOT is a rare entity and behaves similarly to the conventional type, it could show unique clinical and histologic features that may pose problems for differential diagnosis.
Virtual slides
http://www.diagnosticpathology.diagnomx.eu/vs/1979090740113894
doi:10.1186/1746-1596-9-37
PMCID: PMC3932507  PMID: 24555881
Calcifying epithelial odontogenic tumour; Langerhans cell variant; Jaws; Histogenesis; Behaviour
12.  DNA methylation profiling in the thalamus and hippocampus of postnatal malnourished mice, including effects related to long-term potentiation 
BMC Neuroscience  2014;15:31.
Background
DNA methylation has been viewed as the most highly characterized epigenetic mark for genome regulation and development. Postnatal brains appear to exhibit stimulus-induced methylation changes because of factors such as environment, lifestyle, and diet (nutrition). The purpose of this study was to examine how extensively the brain DNA methylome is regulated by nutrition in early life.
Results
By quantifying the total amount of 5-methylcytosine (5mC) in the thalamus and the hippocampus of postnatal malnourished mice and normal mice, we found the two regions showed differences in global DNA methylation status. The methylation level in the thalamus was much higher than that in the hippocampus. Then, we used a next-generation sequencing (NGS)-based method (MSCC) to detect the whole genome methylation of the two regions in malnourished mice and normal mice. Notably, we found that in the thalamus, 500 discriminable variations existed and that approximately 60% were related to neuronal development or psychiatric diseases. Pathway analyses of the corresponding genes highlighted changes for 9 genes related to long-term potentiation (5.3-fold enrichment, P = 0.033).
Conclusions
Our findings may help to indicate the genome-wide DNA methylation status of different brain regions and the effects of malnutrition on brain DNA methylation. The results also indicate that postnatal malnutrition may increase the risk of psychiatric disorders.
doi:10.1186/1471-2202-15-31
PMCID: PMC3941971  PMID: 24555847
Malnutrition; Thalamus; Hippocampus; Mouse model; Global DNA methylation status; Whole genome methylation sequencing; Long-term potentiation; Psychiatric disorders
13.  Deprotonation of D96 in bacteriorhodopsin opens the proton uptake pathway 
Summary
Despite extensive investigation, the precise mechanism controlling the opening of the cytoplasmic proton uptake pathway in Bacteriorhodopsin (bR) has remained a mystery. From an analysis of a novel x-ray structure of the D96G/F171C/F219L triple mutant of bR and 60 independent molecular dynamics simulations of bR photointermediates we report that the deprotonation of D96, a key residue in proton transfer reactions, serves two roles that occur sequentially. First, D96 donates a proton to the Schiff-base. Subsequently, the deprotonation of D96 serves to “unlatch” the cytoplasmic side. The latching function of D96 appears to be remarkably robust, functioning to open hydration channels in all photointermediate structures. These results suggest that the protonation state of D96 may be the critical biophysical cue controlling the opening and closing of the cytoplasmic half-channel in bR. We suspect that this protonation-switch mechanism could also be utilized in other proton pumps to minimize backflow and reinforce directionality.
doi:10.1016/j.str.2012.12.018
PMCID: PMC3642035  PMID: 23394942
bacteriorhodopsin; molecular dynamics; proton pump; conformational change; ion transport; membrane protein; deprotonation
14.  Genome-Wide Linkage, Exome Sequencing and Functional Analyses Identify ABCB6 as the Pathogenic Gene of Dyschromatosis Universalis Hereditaria 
PLoS ONE  2014;9(2):e87250.
Background
As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH.
Methodology
We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation.
Results
Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified ABCB6 as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of ABCB6 in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of ABCB6 in melanocytes and pigmentation. Given the involvement of ABCB6 mutations in coloboma, we performed ophthalmological examination of the DUH carriers of ABCB6 mutations and found ocular abnormalities in them.
Conclusion
Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma.
doi:10.1371/journal.pone.0087250
PMCID: PMC3911924  PMID: 24498303
15.  Three Distinct Modes of Mec1/ATR and Tel1/ATM Activation Illustrate Differential Checkpoint Targeting during Budding Yeast Early Meiosis 
Molecular and Cellular Biology  2013;33(16):3365-3376.
Recombination and synapsis of homologous chromosomes are hallmarks of meiosis in many organisms. Meiotic recombination is initiated by Spo11-induced DNA double-strand breaks (DSBs), whereas chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). Previously, we proposed that budding yeast SC is assembled via noncovalent interactions between the axial SC protein Red1, SUMO chains or conjugates, and the central SC protein Zip1. Incomplete synapsis and unrepaired DNA are monitored by Mec1/Tel1-dependent checkpoint responses that prevent exit from the pachytene stage. Here, our results distinguished three distinct modes of Mec1/Tec1 activation during early meiosis that led to phosphorylation of three targets, histone H2A at S129 (γH2A), Hop1, and Zip1, which are involved, respectively, in DNA replication, the interhomolog recombination and chromosome synapsis checkpoint, and destabilization of homology-independent centromere pairing. γH2A phosphorylation is Red1 independent and occurs prior to Spo11-induced DSBs. DSB- and Red1-dependent Hop1 phosphorylation is activated via interaction of the Red1-SUMO chain/conjugate ensemble with the Ddc1-Rad17-Mec3 (9-1-1) checkpoint complex and the Mre11-Rad50-Xrs2 complex. During SC assembly, Zip1 outcompetes 9-1-1 from the Red1-SUMO chain ensemble to attenuate Hop1 phosphorylation. In contrast, chromosome synapsis cannot attenuate DSB-dependent and Red1-independent Zip1 phosphorylation. These results reveal how DNA replication, DSB repair, and chromosome synapsis are differentially monitored by the meiotic checkpoint network.
doi:10.1128/MCB.00438-13
PMCID: PMC3753904  PMID: 23775120
16.  Neoadjuvant therapy followed by local excision and two-stage total mesorectal excision: a new strategy for sphincter preservation in locally advanced ultra-low rectal cancer 
Gastroenterology Report  2014;2(1):37-43.
Background: With the increased usage of neoadjuvant chemoradiotherapy, improved surgical technique and stapling devices, sphincter-preserving resection has become more frequent for patients with rectal cancer. However, as for locally advanced ultra-low rectal cancer, sphincter-preservation is still facing an enormous challenge.
Objective: To introduce an NLT strategy of sphincter-preservation—neoadjuvant therapy (NT) followed by local excision (LE) and two-stage total mesorectal excision (TME)—into the treatment of locally advanced ultra-low rectal cancer (lesions with anal sphincter invasion).
Methods: From October 2010 to October 2011, nine patients with locally advanced rectal cancer located less than 3 cm from the anal verge were treated by the NLT strategy. All patients had shown good clinical response to NT. The LE procedure was carried transanally 6–8 weeks after completion of the NT. TME was performed to dissect mesorectal lymph nodes 4–6 weeks after LE.
Results: Of the nine patients, the lesion was assessed as T2 in two, T3 in five, and T4 in two before NT, and lymph node metastasis was detected in five patients. The median distance from the tumor to the anal verge was 2.5 cm (range: 1–3 cm). The median follow-up was 27 months (range: 24–34 months). No distant metastasis was detected. Only one patient (11.1%) developed local recurrence at 12 months post-operatively and then underwent abdomino-perineal resection. The remaining eight patients had preserved long-term continence and the median Wexner score at two years post-operation was 4 (range: 2–6).
Conclusion: The new NLT strategy can achieve sphincter-preservation in some patients with ultra-low rectal cancer, with favorable oncological outcome and preservation of normal anal sphincter function.
doi:10.1093/gastro/got040
PMCID: PMC3920994
rectal cancer; sphincter-preservation; neoadjuvant therapy; local excision; total mesorectal excision
17.  Metabolic mapping of deep brain structures and associations with symptomatology in autism spectrum disorders 
Structural neuroimaging studies in autism report atypical volume in deep brain structures which are related to symptomatology. Little is known about metabolic changes in these regions, and how they vary with age and sex, and/or relate to clinical behaviors. Using magnetic resonance spectroscopy we measured N-acetylaspartate, choline, creatine, myoinositol and glutamate in the caudate, putamen, and thalamus of 20 children with autism and 16 typically developing controls (7–18 years). Relative to controls, individuals with autism had elevated glutamate/creatine in the putamen. In addition, both groups showed age-related increases in glutamate in this region. Boys, relative to girls had increased choline/creatine in the thalamus. Lastly, there were correlations between glutamate, choline, and myoinositol in all three regions, and behavioral scores in the ASD group. These findings suggest changes in deep gray matter neurochemistry, which are sensitive to diagnosis, age and sex, and are associated with behavioral differences.
doi:10.1016/j.rasd.2013.10.003
PMCID: PMC3897261  PMID: 24459534
Magnetic resonance spectroscopy; Autism spectrum disorders; Deep gray matter; Caudate nucleus; Putamen; Thalamus and social cognition
18.  A Genome-Wide Association Study Identifies a Locus on TERT for Mean Telomere Length in Han Chinese 
PLoS ONE  2014;9(1):e85043.
Leukocyte telomere length (LTL) is a predictor of aging and a number of age-related diseases. We performed genome-wide association studies of mean LTL in 2632 individuals,with a two-stage replication in 3917 individuals from Chinese populations. To further validate our findings, we get the results of 696 samples from a cohort of European ancestry. We identified two loci associated with LTL that map in telomerase reverse transcriptase (TERT; rs2736100, P = 1.93×10−5) on chromosome 5p15.33 and near keratin 80 (KRT80; rs17653722, P = 6.96×10−6) on 12q13.13. In Chinese population each C allele of rs2736100 and T allele of rs17653722 was associated with a longer mean telomere length of 0.026 and 0.059 T/S, respectively, equivalent to about 3 and 7 years of average age-related telomere attrition. Our findings provide new insights into telomere regulatory mechanism and even pathogenesis of age-related diseases.
doi:10.1371/journal.pone.0085043
PMCID: PMC3897378  PMID: 24465473
19.  Pch2 Prevents Mec1/Tel1-Mediated Hop1 Phosphorylation Occurring Independently of Red1 in Budding Yeast Meiosis 
PLoS ONE  2014;9(1):e85687.
A prominent feature of meiosis in most sexually reproducing organisms is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks are repaired using intact homologous non-sister chromatids rather than sister chromatids. Budding yeast DNA damage checkpoint kinases Mec1 and Tel1 act together with the axial element protein Red1 to promote interhomolog recombination by phosphorylating another axial element protein Hop1. Mec1 and Tel1 also phosphorylate γH2A and the synaptonemal complex protein Zip1 independently of Red1 to facilitate premeiotic DNA replication and to destabilize homology-independent centromere pairing, respectively. It has been unclear why Hop1 phosphorylation is Red1-dependent. Here, we report that the pachytene checkpoint protein 2 (Pch2) specifically prevents Red1-independent Hop1 phosphorylation. Our findings reveal a new function for Pch2 in linking two axial element proteins Red1 and Hop1 thus coordinating their effects in meiotic recombination and the checkpoint network.
doi:10.1371/journal.pone.0085687
PMCID: PMC3897485  PMID: 24465650
20.  The Gate that Governs Sulfotransferase Selectivity 
Biochemistry  2012;52(2):415-424.
Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl-moiety (-SO3) from activated sulfate (3′-phosphoadenosine 5′-phosphosulfate, PAPS) to the hydroxyls and primary amines of numerous metabolites, drugs and xenobiotics. Receipt of the sulfuryl-group often radically alters acceptor-target interactions. How these enzymes select particular substrates from the hundreds of candidates in a complex cytosol remains an important question. Recent work reveals PAPS binding causes SULT2A1to undergo an isomerization that controls selectivity by constricting the opening through which acceptors must pass to enter the active site. The enzyme maintains an affinity for large substrates by isomerizing between the open and closed states with nucleotide bound. Here, the molecular basis of the nucleotide-induced closure is explored in equilibrium and non-equilibrium molecular dynamics simulations. The simulations predict that the active-site “cap,” which covers both the nucleotide and acceptor binding sites, opens and closes in response to nucleotide. The cap subdivides into nucleotide and acceptor halves whose motions, while coupled, exhibit an independence that can explain the isomerization. In-silico weakening of electrostatic interactions between the cap and base of the active site causes the acceptor-half of the cap to open and close while the nucleotide lid remains shut. Simulations predict that SULT1A1, the most abundant SULT in human liver, will utilize a similar selection mechanism. This prediction is tested using fulvestrant, an antiestrogen too large to pass through the closed pore, and estradiol, which is not restricted by closure. Equilibrium and presteady state binding studies confirm that SULT1A1 undergoes a nucleotide induced isomerzation that controls substrate selection.
doi:10.1021/bi301492j
PMCID: PMC3549632  PMID: 23256751
sulfotransferase; SULT; SULT1A1; SULT2A1; fulvestrant; estradiol; kinetic; mechanism; ligand; binding; presteady; fluorescence; structure; selection; substrate; GROMACS; molecular dynamics
21.  DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape 
Nature genetics  2013;45(7):10.1038/ng.2649.
Introduction
Transposable element (TE) derived sequences comprise half of our genome and DNA methylome, and are presumed densely methylated and inactive. Examination of the genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues revealed unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the tissue type and their expression correlated strongly with hypomethylation of the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks including H3K4me1 and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and exhibited evidence for evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type-specific regulatory networks, and have acquired tissue-specific epigenetic regulation.
doi:10.1038/ng.2649
PMCID: PMC3695047  PMID: 23708189
22.  Evodiamine Inhibits Insulin-Stimulated mTOR-S6K Activation and IRS1 Serine Phosphorylation in Adipocytes and Improves Glucose Tolerance in Obese/Diabetic Mice 
PLoS ONE  2013;8(12):e83264.
Evodiamine, an alkaloid extracted from the dried unripe fruit of the tree Evodia rutaecarpa Bentham (Rutaceae), reduces obesity and insulin resistance in obese/diabetic mice; however, the mechanism underlying the effect of evodiamine on insulin resistance is unknown. This study investigated the effect of evodiamine on signal transduction relating to insulin resistance using obese/diabetic KK-Ay mice and an in vitro adipocyte culture. There is a significant decrease in the mammalian target of rapamycin (mTOR) and ribosomal S6 protein kinase (S6K) signaling in white adipose tissue (WAT) in KK-Ay mice treated with evodiamine, in which glucose tolerance is improved. In addition, reduction of insulin receptor substrate 1 (IRS1) serine phosphorylation, an indicator of insulin resistance, was detected in their WAT, suggesting suppression of the negative feedback loop from S6K to IRS1. As well as the stimulation of IRS1 and Akt serine phosphorylation, insulin-stimulated phosphorylation of mTOR and S6K is time-dependent in 3T3-L1 adipocytes, whereas evodiamine does not affect their phosphorylation except for an inhibitory effect on mTOR phosphorylation. Moreover, evodiamine inhibits the insulin-stimulated phosphorylation of mTOR and S6K, leading to down-regulation of IRS1 serine phosphorylation in the adipocytes. Evodiamine also stimulates phosphorylation of AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, which may cause down-regulation of mTOR signaling in adipocytes. A similar effect on AMPK, mTOR and IRS1 phosphorylation was found in adipocytes treated with rosiglitazone. These results suggest evodiamine improves glucose tolerance and prevents the progress of insulin resistance associated with obese/diabetic states, at least in part, through inhibition of mTOR-S6K signaling and IRS1 serine phosphorylation in adipocytes.
doi:10.1371/journal.pone.0083264
PMCID: PMC3877036  PMID: 24391749
23.  De novo Sequencing, Characterization, and Comparison of Inflorescence Transcriptomes of Cornus canadensis and C. florida (Cornaceae) 
PLoS ONE  2013;8(12):e82674.
Background
Transcriptome sequencing analysis is a powerful tool in molecular genetics and evolutionary biology. Here we report the results of de novo 454 sequencing, characterization, and comparison of inflorescence transcriptomes of two closely related dogwood species, Cornus canadensis and C. florida (Cornaceae). Our goals were to build a preliminary source of genome sequence data, and to identify genes potentially expressed differentially between the inflorescence transcriptomes for these important horticultural species.
Results
The sequencing of cDNAs from inflorescence buds of C. canadensis (cc) and C. florida (cf), and normalized cDNAs from leaves of C. canadensis resulted in 251799 (ccBud), 96245 (ccLeaf) and 114648 (cfBud) raw reads, respectively. The de novo assembly of the high quality (HQ) reads resulted in 36088, 17802 and 21210 unigenes for ccBud, ccLeaf and cfBud. A reference transcriptome for C. canadensis was built by assembling HQ reads of ccBud and ccLeaf, containing 40884 unigenes. Reference mapping and comparative analyses found 10926 sequences were putatively specific to ccBud, and 6979 putatively specific to cfBud. Putative differentially expressed genes between ccBud and cfBud that are related to flower development and/or stress response were identified among 7718 shared sequences by ccBud and cfBud. Bi-directional BLAST found 87 (41.83% of 208) of Arabidopsis genes related to inflorescence development had putative orthologs in the dogwood transcriptomes. Comparisons of the shared sequences by ccBud and cfBud yielded 65931 high quality SNPs between two species. The twenty unigenes with the most SNPs are listed as potential genetic markers for evolutionary studies.
Conclusions
The data provide an important, although preliminary, information platform for functional genomics and evolutionary developmental biology in Cornus. The study identified putative candidates potentially involved in the genetic regulation of inflorescence evolution and/or disease resistance in dogwoods for future analyses. Results of the study also provide markers useful for dogwood phylogenomic studies.
doi:10.1371/journal.pone.0082674
PMCID: PMC3873919  PMID: 24386108
24.  Effects of Understory Vegetation and Litter on Plant Nitrogen (N), Phosphorus (P), N∶P Ratio and Their Relationships with Growth Rate of Indigenous Seedlings in Subtropical Plantations 
PLoS ONE  2013;8(12):e84130.
Establishing seedlings in subtropical plantations is very important for forest health, succession and management. Information on seedling nutrient concentrations is essential for both the selection of suitable indigenous tree species to accelerate succession of the established plantation and sustainable forest management. In this study, we investigated the concentrations of nitrogen ([N]), phosphorus ([P]), and N∶P ratio in leaves, stems and roots of seedlings of three indigenous tree species (Castanopsis chinensis, Michelia chapensis and Psychotria rubra) transplanted with removing or retaining understory vegetation and litter at two typical subtropical forest plantations (Eucalyptus plantation and native species plantation). We also measured the relative growth rate (RGR) of seedling height, and developed the relationships between RGR and leaf [N], [P] and N∶P ratio. Results showed that treatments of understory vegetation and associated litter (i.e. removal or retained) generally had no significant effects on leaf [N], [P], N∶P ratio and RGR of the transplanted tree seedlings for the experimental period. But among different species, there were significant differences in nutrient concentrations. M. chapensis and P. rubra had higher [N] and [P] compared to C. chinensis. [N] and [P] also varied among different plant tissues with much higher values in leaves than in roots for all indigenous species. RGR of indigenous tree seedlings was mostly positively correlated with leaf [N] and [P], but negatively correlated with leaf N∶P ratio. Considering the low [P] and high N∶P ratio observed in the introduced indigenous tree seedlings, we propose that the current experimental plantations might be P limited for plant growth.
doi:10.1371/journal.pone.0084130
PMCID: PMC3873957  PMID: 24386340
25.  Empirical Evidence Supporting Frequent Cryptic Speciation in Epiphyllous Liverworts: A Case Study of the Cololejeunea lanciloba Complex 
PLoS ONE  2013;8(12):e84124.
Cryptic species are frequently recovered in plant lineages, and considered an important cause for divergent of morphological disparity and species diversity. The identification of cryptic species has important implications for the assessment of conservation needs of species aggregates. The mechanisms and processes of the origin of cryptic species diversity are still poorly understand based on the lack of studies especially in context of environment factors. Here we explored evidence for cryptic species within the epiphyllous liverworts Cololejeunea lanciloba complex based on two loci, the plastid trnL-F region and the nuclear ribosomal ITS region. Several analytic approaches were employed to delimit species based on DNA sequence variation including phylogenetic reconstruction, statistical parsimony networks analysis and two recently introduced species delimitation criteria: Rosenberg’s reciprocal monophyly and Rodrigo’s randomly distinct. We found evidence for thirteen genetically distinct putative species, each consisting of more than one haplotype, rather than four morphologically-circumscribed species. The results implied that the highly conserved phenotypes are not congruent with the genetic differentiation, contributing to incorrect assessments of the biodiversity of epiphyllous liverworts. We hypothesize that evolution of cryptic species recovered may be caused by selection of traits critical to the survival in epiphyllous habitats combined with limited developmental options designed in the small body.
doi:10.1371/journal.pone.0084124
PMCID: PMC3867491  PMID: 24367634

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